Adenoviruses (Ads) are common human viruses that cause mainly respiratory infections and have great potential for vector-based gene delivery and vaccination strategies. Ads provide numerous advantages for vector design, including the fact that they have been well studied as a model system in cell and molecular biology and we have a good understanding of their life cycle. In the previous funding period, we made exciting new discoveries related to the role of fiber flexibility in receptor usage and cell entry; determined a 6[unreadable] resolution cryoEM structure of an Ad vector;localized the membrane-lytic protein VI and protein IX, which has been used as a retargeting platform, within the Ad capsid;identified likely binding sites on hexon for the human blood coagulation Factor X, which plays an important role in targeting Ad to the liver;investigated the nature of the cell entry defect of the temperature sensitive mutant Ad2ts1;and shown that [unreadable]v integrins undergo significant conformational change after binding to Ad. We have also developed a script assisted microscopy package, which facilitates semi-automated cryoEM data acquisition, and collaborated on the development of cryoEM guided de novo protein fold elucidation methods. The specific aims of this proposal are to determine high resolution cryoEM structures of: 1) Ad complexed with human defensin 5, a component of the innate immune system with both antimicrobial and antiviral activity;2) Ad complexed with Factor X, to determine the key residues of hexon that are involved in the interaction and might be mutated to ablate Factor X binding and liver targeting;and 3) the multiprotein complex that binds the Ad DNA packaging sequence and is critical for Ad assembly. Cryoelectron tomography, combined with antibody labeling, will be used to locate the DNA packaging complex within the Ad virion. In addition, we will perform non-icosahedral averaging on the protein VI density in the Ad capsid to facilitate atomic modeling of this membrane-lytic factor;and apply cryoEM guided de novo methods to enhance structure determination of the Ad capsid and the Ad- integrin receptor complex. These studies will contribute to a greater understanding of the Ad life cycle including key interactions with cell receptors, antimicrobial peptides, and tissue targeting factors in the blood. This knowledge may in turn lead to novel strategies for antiviral drug design, Ad vector retargeting, and shielding of Ad vectors from the host immune system. The long term goal of these studies is to provide structural information that will help guide the redirection of Ad for numerous potential biomedical applications.